TILLING

An alternative to standard mammalian gene targeting has recently yielded
the first mutant fish by a reverse genetics approach. This approach, which is also known as TILLING (Targeting Induced Local Lesions In Genomes), had been pioneered in other organisms such as Arabidopsis. The method consists of identifying F1 males from standard ethylnitrosourea (ENU) screens that harbor heterozygous mutations in the gene of interest (see Figure). For this, a library of DNA from 2,000-4,000 F1 males is generated in parallel with a library of matching cryopreserved sperm samples. Genomic DNA from the sperm donors is arrayed for high-throughput mutation screening by PCR and sequencing of the exons of the gene of interest. Alternatively, pooled DNA from 4-8 donors can be screened for mutations using the CelI endonuclease. Individuals from positive pools are then sequenced to identify the nucleotide change. Lines of fish carrying an inactivating mutation are then established by thawing the corresponding sperm samples and performing in vitro fertilization. Inbreeding of heterozygous offspring will produce homozygous mutants for the gene of interest, and are then available for phenotypic analysis, enhancer- and suppressor screens.